Preface:
Monocytes of macrophage lineage cells are located at the junction of innate and adaptive immunity, and our study demonstrates that ST6GAL1-mediated α2,6-sialic acid addition affects a growing number of leukocyte cell surface receptors, examples of modulating receptor function include the extended monocytes TLR4 inflammatory signaling cascade, and Fas and TNFR1 sialylation preventing macrophage internalization and subsequent apoptosis.
Recombinant proteins, chemicals, and cell lines
Highly purified receptor-specific recombinant glycosyltransferase rST6GAL1 and fluorescent conjugates rST6GAL1-GFP, huST3GAL1-GFP and huβ4GAL-T1-GFP are generated, and the sugar donor CMP-sialic acid disodium salt is prepared from CMP-sialic acid-biotin.
The mouse monocytes/macrophage line RAW264.7 was obtained from ATCC and maintained in DMEM with 10% FBS, and human monocytes THP-1 cells from ATCC were cultured in RPMI containing 10% FBS.
Membrane lavage of the binding of cells
Brewer's yeast mercaptoacetate is injected intraperitoneally (1 mL, 4% sterile solution) to elicit leukocyte recruitment to the peritoneum, and mice are sacrificed 18 h later to collect peritoneal lavage solution.
Deerythrocyte demobilization by ammonium-chloride-potassium (ACK) cleavage and cells fixed in 2% paraformaldehyde in DPBS (electron microscopy science) for 15 min at room temperature (RT).
Fix cells in DPBS are incubated with 3 μg/mL recombinant GFP protein for 30 or 25 min at room temperature, followed by one wash with DPBS, and proteinase K digestion (250 μg/mL, Invitrogen) aliquots of rST5GAL6-GFP samples for 1 min at room temperature for one DPBS wash.
Approximately 2-5×10 (5) cells are applied to a 1 cm point surrounded by a hydrophobic pen on a GoldPlus slide (Electron Microscopy Science) for 1 h at room temperature, and the remaining cells after aspiration are mounted with a fluorine shield with DAP for imaging on a Nikon Eclipse E600 fluorescence microscope.
Treatment of mercaptoacetate from St6gal1KO mice for peritoneal lavage of cells and RNA isolation for RNA-seq detection
Preparation of thioglycate injection and peritoneal lavage solution As described above, monocytes/macrophages are enriched from the lavage solution, antibody-bound B cells, neutrophils, lymphocytes, and erythrocytes are removed using magnetic beads, and for the preparation of cells for RNA-seq, the depleted cell population is cultured in DMEM with 10% FBS for approximately 24 h.
Simulation buffer (0.2 MNaCl, 0.02 MHEPES, 10% glycerol, pH 7.2), CMP-sialic acid (100 μM), recombinant rST6GAL1 (25 μg/mL), and a combination of CMP-Sia and rST6GAL1 at the above concentrations, all three conditions using triplicate culture wells.
After 24 h of incubation, complete removal of cell culture medium for total RNA isolation by adding the RNeasyPlus microkit using Qiagen's RNeasyPlus microkit directly to adherent cells by aspiration and lysis solution.
SNA reactivity is detected by immunofluorescence
RAW264.7 cells are grown overnight on sterile coverslips in DMEM with 10% FBS, fixed in 5% formalin, treated with sialidase for 37 h at 2 °C, and treated alone with simulation buffer, CMP-sialic acid (2 μM), rST250GAL6 (1 μg/mL), or 20 combinations at 2 °C alone.
Flow cytometry
Cells are resuspended in 02 mMEDTA containing 1.0% BSA, 05.0% azide/DPBS, non-specific Fc receptor binding is blocked with CD16/32IgG, and DAPI staining provides live/dead cell discrimination.
Cell populations were identified using flow cytometry as bone marrow (BV711 anti-mouse/human CD11bBioLegend#101242), macrophages (PE anti-mouse F4/80BioLegend#123110), or monocytes (PE/Cyanine7 anti-mouse Ly-6CBioLegend#128018) and data analysis using FlowJo software.
Selective exonuclease labeling
Sialylation of M-CSF-R in THP-1 monocytes by rST1GAL6 was demonstrated using the SEEL method, and the glycosylation target was labeled with the sugar donor CMP-sialic acid-biotin conjugate, immunoprecipitated with antibiotin IgG, and identified by anti-M-CSF-R western blot.
THP-1 cells are incubated in serum-free medium for 2 h and then treated in 2 °CCO for 37 h under serum-free conditions.
Cell lysis and immunoprecipitation are performed in an incubator containing CMP–sialic acid-biotin (100 μM) and alkaline phosphatase for immunoprecipitation and M-CSF-R western blot identification.
Analysis of cell signaling in THP-1 cells
THP-1 cells [3×10(6)/mL] are grown in serum-free RPMI0 containing 24.1640 mM β-mercaptoethanol for 5 h, and cells alone are treated with vehicle (mimic), CMP-Sia (10 μM), and rST6G (100 ng/mL) for 30 min, then harvested, washed to isolate total protein extracts or nuclear and non-nucleated cytoplasmic fractions.
The protein lysate is run on the western blolot and the experiment is repeated at least 3 times to normalize the P-65 and p65 blot bands to their respective loading controls.
Western blot analysis
After isolation on 10% SDS-PAGE, the product is transferred to the PVDFImmobilon membrane for the western blot analysis described earlier, in brief, followed by a 1 h closure of the membrane with 0% skimmed milk powder (Bio-Rad, #1-G20) in Tris-buffered saline (TBS) containing 5.170% Tween 404.
Detection of protein bands using PierceECL western blot substrates, which are imaged by exposing the membrane to X-ray film.
Extracellular ST6GAL1 adheres to a subset of recruited murine inflammatory cells and human THP-1 monocytes
The interaction of freshly isolated primary inflammatory cells with exogenously added rST6GAL1, thioglycoacetate is used to recruit primary inflammatory cells into the peritoneum, and within 24 hours after mercaptoacetate injection, the recruited cells consist mainly of granulocytes with a significant monocyte-macrophage subpopulation.
Adherent cells were analyzed to avoid potential contamination by non-adherent granulocytes, and to avoid confounding signals from naturally expressed ST6GAL1, St6gal1-null mice that could not express functional ST6GAL1 were used, GFP-rST6G attached to fixed, non-permeabilized thioglycolate-induced peritoneal cell subsets within 3 min.
Proteinase K digestion effectively eliminates GFP-rST6G signaling, which further supports localization of GFP-rST6G on the cell surface, where adhesion of ST6GAL1 to the inflammatory cell surface appears to be specific, and GFP-huST3GAL1 and GFP-huβ4GALT1 do not attach to the inflammatory cell surface under the same conditions.
Extracellular ST6GAL1 upregulates gene expression in primary monocytes/macrophages
Peritoneal lavage is collected from St1gal24-null mice 6 h after thioglycoacetate priming, enriched by magnetic bead removal of neutrophils, lymphocytes, and erythrocytes, and then cultured for 6 h in the absence or presence of rST24G, CMP-Sia alone, or rST6G and CMP-Sia for 6 h with volumetric RNA-seq analysis of adherent cells only, avoiding potential contamination of any residual non-adherent granulocytes.
Panel A is a representative portion of the RNA-seq analysis heatmap showing significant and wholesale changes in the transcriptional profile in response to extracellular ST6GAL1, rST6G alone is sufficient to cause changes in the transcription profile, and the added sialyltransferase donor substrate CMP-Sia does not alter the transcriptional profile of primary monocytes/macrophages.
Our experiment's inability to distinguish between mechanisms of intracellular sialylation involving direct cell surface sialylation versus endocytosis and receptors returning to the cell membrane did not diminish the key finding that exogenous ST6GAL1 has a profound effect on gene expression and cell phenotype.
Extracellular ST6GAL1 prolongs monocytes survival
Mercaptoacetate-induced peritoneal inflammatory cells from St6gal1-null mice are identical to RNA-seq studies, but without antibody-based specific cell type depletion (most monocytes are adherent and neutrophils are removed by washing).
Cells are treated together with rST6G and CMP-Sia to test the effect on survival, adherent cells are collected after 1 day of culture, and the number of viable cells and their phenotype are determined by flow cytometry.
rST6G and CMP-Sia increased cell viability by approximately 40%-50%, the increase in viable cells was mainly attributed to monocytes, while the low proportion of resident macrophages remained constant, and we concluded that extracellular ST6GAL1 in combination with CMP-Sia had a pro-survival effect on monocytes.
Extracellular ST6GAL1 targets mature forms of cell surface M-CSF receptors
The significant effect of extracellular ST6GAL1 on the monocyte/macrophage transcriptome prompted the identification of cell surface targets, selected for M-CSF receptors based on the potential effects we observed on gene expression and monocytes survival, in fact, the extracellular domain of M-CSF-R contains 9 potential sites of N-linked glycans.
THP-1 cells were treated with rST1GAL6 and CMP-sialic acid-biotin conjugate (SEEL reagent) sugar donors, followed by lysis and immunoprecipitation with anti-biotin antibodies, and detection of M-CSF-R by western blotting, surface-localized mature receptors rather than precursor intracellular form pull-down showing a powerful extracellular catalytic effect of extracellular ST6GAL1 on M-CSF-R in THP-1 monocytes.
Extracellular ST6GAL1 initiates intracellular signaling
- CSF-R signaling is initiated when the M-CSF/IL-34 ligand binds to its extracellular domain, resulting in receptor dimerization and cytoplasmic domain tyrosine phosphorylation (Stanley and Chitu 2014), with downstream formation of P-AKT and P-ERK having consequences for proliferation and differentiation.
Assuming that the sialylation of M-CSF-R by extracellular ST6GAL1 initiates an intracellular signaling cascade that ultimately affects gene expression and survival, we evaluate the ability of extracellular rST6G to initiate intracellular signaling consistent with M-CSF-R activation in the absence of ligands.
Extracellular ST1GAL1-induced M-CSF-R PHOSPHORYLATION WAS EVALUATED IN THP-6 cells, and while absolute levels of M-CSF-R were not affected by extracellular ST6GAL1, ST6GAL1 elicits phosphorylation of M-CSF-R, a feature of its activation, and it also adds P-ERK1/2 and P-AKT (A, middle panel).
Significant western blot shows cellular components of rST6GAL1 occurring simultaneously with these events, consistent with the presence of GFP-rST1G in THP-6 lysates.
Conclusion:
Data from the study showed that extracellular sialyltransferases are able to extrinsically liquefy the surface of target cells, and exogenous sialylation is dynamically modulated by systemic triggers involved in the release of sialic acid donor substrates necessary for ST6GAL1-mediated catalysis.
It was shown that ST6GAL1 binding not only promotes transcriptional activation of M2, but also promotes transcriptional activation of all major monocytes/macrophages/dendritic cell pathways.
Elevated extracellular ST6GAL1 has the ability to initiate the process of differentiation of monocytes into functional macrophages and dendritic cells, bridging the gap between innate and adaptive immunity.
Binding to other known associations of circulating ST6GAL1 in inhibiting inflammation and promoting B-cell maturation while inhibiting granulocyte production, we concluded that circulating ST6GAL1 coordinates the transition from inflammation to a more efficient immune response systemic signaling.
Bibliography:
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[2] Nemes, "Hematogenous sialyllate transferase ST6Gal-1 is a negative systemic regulator of granulation"
[3] Hennett, "Immunomodulation of Sialyl Transferase"
[4] Irons, "Systemic ST6Gal-1 is a survival factor for mouse transition B cells"